Viral vectors as part of an integrated functional genomics program.

Over the past decade, viral vectors have slowly gained mainstream acceptance in the neuroscience and genetics communities for the in vivo study of gene function [1]. Using stereotactic techniques, it is possible to characterize neuroanatomical relationships through the delivery of neurotropic viral vectors to specific brain regions. More sophisticated studies combine viral vectors with other methods of genetic manipulation such as germline transgenic mice. As more is learned about the properties of different viral vectors, it has become possible to use viral vectors to test hypotheses about the function of genes, through targeted in vivo delivery to the central nervous system (CNS). The effects of gene expression in the brain can be measured on the molecular, biochemical, electrophysiological, morphological, and behavioral levels. We propose that viral vectors should be considered as part of an integrated functional genomics platform in the CNS.     

Morphological abnormalities, neonatal mortality, and reproductive abnormalities in mice transgenic for diphtheria toxin genes that are driven by the promoter for adipocyte lipid binding protein.

Transgenic mice were used in an experiment that was designed to serve as a model of a possible approach to reducing the amount of carcass fat in meat animals. The objective was to reduce the number of adipocytes in transgenic mice thereby restricting the capacity to accumulate lipid. Our approach employed the technique of genetic ablation. The promoter for the adipocyte lipid binding protein gene was used in an attempt to direct expression of diphtheria toxin genes specifically to adipocytes. Three diphtheria toxin genes were used; they encode, respectively, an extremely cytotoxic wild type toxin, a less toxic attenuated toxin, and a nonfunctional toxin. While it was not possible to accurately assess effects of the transgenes on lipid accumulation, several informative observations were noted. A large percentage of transgenic founder mice that harbor either wild type or attenuated toxin genes are morphologically abnormal, die as neonates, or exhibit reproductive abnormalities including sterility or failure to transmit the transgene to offspring. In contrast, mice that harbor the nonfunctional toxin gene or are nontransgenic rarely have these same abnormalities. These results suggest that the transgenic mice are expressing the transgenes in cells other than adipocytes and that the aberrant production of functional toxin is responsible for the congenital abnormalities. The production of morphological and reproductive abnormalities in transgenic animals should be useful for investigating normal developmental processes.     

Morphological abnormalities,neonatal mortality,and reproductive abnormalities in mice transgenic for diphtheria toxin genes that are driven by the promoter for adipocyte lipid binding protein

Transgenic mice were used in an experiment that was designed to serve as a model of a possible approach to reducing the amount of carcass fat in meat animals. The objective was to reduce the number of adipocytes in transgenic mice thereby restricting the capacity to accumulate lipid. Our approach employed the technique of genetic ablation. The promoter for the adipocyte lipid binding protein gene was used in an attempt to direct expression of diphtheria toxin genes specifically to adipocytes. Three diphtheria toxin genes were used; they encode, respectively, an extremely cytotoxic wild type toxin, a less toxic attenuated toxin, and a nonfunctional toxin. While it was not possible to accurately assess effects of the transgenes on lipid accumulation, several informative observations were noted. A large percentage of transgenic founder mice that harbor either wild type or attenuated toxin genes are morphologically abnormal, die as neonates, or exhibit reproductive abnormalities including sterility or failure to transmit the transgene to offspring. In contrast, mice that harbor the nonfunctional toxin gene or are nontransgenic rarely have these same abnormalities. These results suggest that the trans-genic mice are expressing the transgenes in cells other than adipocytes and that the aberrant production of functional toxin is responsible for the congenital abnormalities. The production of morphological and reproductive abnormalities in transgenic animals should be useful for investigating normal developmental processes.     

Recent advances in the manipulation of murine gene expression and its utility for the study of human neurological disease

Transgenic mouse models have vastly contributed to our knowledge of the genetic and molecular pathways underlying the pathogenesis of neurological disorders that affect millions of people worldwide. Not only have they allowed the generation of disease models mimicking the human pathological state but they have also permitted the exploration of the pathological role of specific genes through the generation of knock-out and knock-in models. Classical constitutive transgenic mice have several limitations however, due to behavioral adaptation process occurring and conditional mouse models are time-consuming and often lack extensive spatial or temporal control of gene manipulation. These limitations could be overcome by means of innovative methods that are now available such as RNAi, viral vectors and large cloning DNA vectors. These tools have been extensively used for the generation of mouse models and are characterized by the superior control of transgene expression that has been proven invaluable in the assessment of novel treatments for neurological diseases and to further investigate the molecular processes underlying the etiopathology of neurological disorders. Furthermore, in association with classical transgenic mouse models, they have allowed the validation of innovative therapeutic strategies for the treatment of human neurological disorders. This review describes how these tools have overcome the limitations of classical transgenic mouse models and how they have been of value for the study of human neurological diseases.     

Elevated Neuroglobin Lessens Neuroinflammation and Alleviates Neurobehavioral Deficits Induced by Acute Inhalation of Combustion Smoke in the Mouse

Acute inhalation of combustion smoke produces long-term neurologic deficits in survivors. To study the mechanisms that contribute to the development of neurologic deficits and identify targets for prevention, we developed a mouse model of acute inhalation of combustion smoke, which supports longitudinal investigation of mechanisms that underlie the smoke induced inimical sequelae in the brain. Using a transgenic mouse engineered to overexpress neuroglobin, a neuroprotective oxygen-binding globin protein, we previously demonstrated that elevated neuroglobin preserves mitochondrial respiration and attenuates formation of oxidative DNA damage in the mouse brain after smoke exposure. In the current study, we show that elevated neuronal neuroglobin attenuates the persistent inflammatory changes induced by smoke exposure in the mouse brain and mitigates concordant smoke-induced long-term neurobehavioral deficits. Specifically, we found that increases in hippocampal density of GFAP and Iba-1 positive cells that are detected post-smoke in wild-type mice are absent in the neuroglobin overexpressing transgenic (Ngb-tg) mice. Similarly, the smoke induced hippocampal myelin depletion is not observed in the Ngb-tg mice. Importantly, elevated neuroglobin alleviates behavioral and memory deficits that develop after acute smoke inhalation in the wild-type mice. Taken together, our findings suggest that the protective effects exerted by neuroglobin in the brains of smoke exposed mice afford protection from long-term neurologic sequelae of acute inhalation of combustion smoke. Our transgenic mouse provides a tool for assessing the potential of elevated neuroglobin as possible strategy for management of smoke inhalation injury.

     

Simple Databases to Monitor the Generation and Organisation of Transgenic Mouse Colonies

The generation and analysis of transgenic mice has become an important tool to progress our understanding of human and mouse gene function and its association with human genetic diseases. Animal models, based on genetically modified mice, both standard transgenic and knock-out animals, are increasingly being used world-wide. Monitoring of transgenic mouse production and transgenic mouse colonies is required to efficiently manage the resources that are available. Here, I describe three independent FileMaker databases (transgenics, mymouse and cages) that have been developed to track the generation of transgenic mice, the organisation of transgenic mouse colonies and the distribution of mice in cages. These three databases are freely available for academic use.     

Age- and region-specific imbalances of basal amino acids and monoamine metabolism in limbic regions of female Fmr1 knock-out mice

The Fragile X syndrome, a common form of mental retardation in humans, originates from the loss of expression of the Fragile X mental retardation gene leading to the absence of the encoded Fragile X mental retardation protein 1 (FMRP). A broad pattern of morphological and behavioral abnormalities is well described for affected humans as well as Fmr1 knock-out mice, a transgenic animal model for the human Fragile X syndrome. In the present study, we examined neurochemical differences between female Fmr1 knock-out and wildtype mice with particular focus on neurotransmission. Significant age- and region-specific differences of basal tissue neurotransmitter and metabolite levels measured by high performance liquid chromatography were found. Those differences were more numerous in juvenile animals (postnatal day (PND) 28-31) compared to adults (postnatal day 209-221). In juvenile female knock-out mice, especially aspartate and taurine were increased in cortical regions, striatum, cerebellum, and brainstem. Furthermore, compared to the wildtype animals, the juvenile knock-out mice displayed an increased level of neuronal inhibition in the hippocampus and brainstem reflected by decreased ratios of (aspartate + glutamate)/(taurine + GABA), as well as an increased dopamine (DA) turnover in cortical regions, striatum, and hippocampus. These results provide the first evidence that the lack of FMRP expression in female Fmr1 knock-out mice is accompanied by age-dependent, region-specific alterations in brain amino acids, and monoamine turnover, which might be related to the reported synaptical and behavioural alterations in these animals.     

Functional Deficiency of MHC Class I Enhances LTP and Abolishes LTD in the Nucleus Accumbens of Mice

Major histocompatibility complex class I (MHCI) molecules were recently identified as novel regulators of synaptic plasticity. These molecules are expressed in various brain areas, especially in regions undergoing activity-dependent synaptic plasticity, but their role in the nucleus accumbens (NAc) is unknown. In this study, we investigated the effects of genetic disruption of MHCI function, through deletion of β2-microblobulin, which causes lack of cell surface expression of MHCI. First, we confirmed that MHCI molecules are expressed in the NAc core in wild-type mice. Second, we performed electrophysiological recordings with NAc core slices from wild-type and β2-microglobulin knock-out mice lacking cell surface expression of MHCI. We found that low frequency stimulation induced long-term depression in wild-type but not knock-out mice, whereas high frequency stimulation induced long-term potentiation in both genotypes, with a larger magnitude in knock-out mice. Furthermore, we demonstrated that knock-out mice showed more persistent behavioral sensitization to cocaine, which is a NAc-related behavior. Using this model, we analyzed the density of total AMPA receptors and their subunits GluR1 and GluR2 in the NAc core, by SDS-digested freeze-fracture replica labeling. After repeated cocaine exposure, the density of GluR1 was increased, but there was no change in total AMPA receptors and GluR2 levels in wild-type mice. In contrast, following repeated cocaine exposure, increased densities of total AMPA receptors, GluR1 and GluR2 were observed in knock-out mice. These results indicate that functional deficiency of MHCI enhances synaptic potentiation, induced by electrical and pharmacological stimulation.     

Behavioral assessment of Alzheimer's transgenic mice following long-term Abeta vaccination: task specificity and correlations between Abeta deposition and spatial memory.

Long-term vaccinations with human beta-amyloid peptide 1-42 (Abeta1-42) have recently been shown to prevent or markedly reduce Abeta deposition in the PDAPP transgenic model of Alzheimer's disease (AD). Using a similar protocol to vaccinate 7.5-month-old APP (Tg2576) and APP+PS1 transgenic mice over an 8-month period, we previously reported modest reductions in brain Abeta deposition at 16 months. In these same mice, Abeta vaccinations had no deleterious behavioral effects and, in fact, benefited the mice by providing partial protection from age-related deficits in spatial working memory in the radial arm water maze task (RAWM) at 15.5 months. By contrast, control-vaccinated transgenic mice exhibited impaired performance throughout the entire RAWM test period at 15.5 months. The present study expands on our initial report by presenting additional behavioral results following long-term Abeta vaccination, as well as correlational analyses between cognitive performance and Abeta deposition in vaccinated animals. We report that 8 months of Abeta vaccinations did not reverse an early-onset balance beam impairment in transgenic mice. Additionally, in Y-maze testing at 16 months, all mice showed comparable spontaneous alternation irrespective of genotype or vaccination status. Strong correlations were nonetheless present between RAWM performance and extent of "compact" Abeta deposition in both the hippocampus and the frontal cortex of vaccinated APP+PS1 mice. Our results suggest that the behavioral protection of long-term Abeta vaccinations is task specific, with preservation of hippocampal-associated working memory tasks most likely to occur. In view of the early short-term memory deficits exhibited by AD patients, Abeta vaccination of presymptomatic AD patients could be an effective therapeutic to protect against such cognitive impairments.     

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.     

Early Changes in Behavior Deficits, Amyloid β-42 Deposits and MAPK Activation in Doubly Transgenic Mice Co-expressing NSE-Controlled Human Mutant PS2 and APPsw

1. Doubly transgenic mice were some differences in the period proceeding of the development of Abeta-42 deposits and behavioral deficits. It was not characterized human mutant PS2 (hPS2) with APPsw in the brains of double transgenic mice. The aim of this study was to examine whether doubly transgenic mice co-expressing NSE-controlled APPsw and hPS2m develop AD-like phenotypes much earlier than singly APPsw or hPS2m alone. 2. We produced doubly transgenic mice from a cross between our previously created NSE-controlled hPS2m and an APPsw transgenic line. This doubly transgenic line was quantitatively produced by cross with age-matched control mice, and the produced mice were separated into 5, 6, 7 and 8-month old age groups. At the age of 8 months, the four groups of mice were tested for behavioral function, levels of Abeta-42 deposition, and potential signaling events. 3. It was shown that all the AD-like phenotypes, including behavior deficits, Abeta-42 levels, MAPK activation and ER expressions in doubly transgenic mice develop much earlier in the early time of AD development than their singly transgenic and non-transgenic littermates. 4. The results suggest that elevated Abeta-42 levels, and MAPK activation in doubly transgenic mice are model for early diagnosis and treatment of AD with therapeutic drug.     

Igf2r improves the survival and transmission ratio of Igf2 transgenic mice

Mammals with excess insulin-like growth factor 2 (IGFII) during embryogenesis have developmental defects that can lead to perinatal lethality. In adults, higher levels of IGFII increase the risk of cancer and may accelerate the development of atherosclerosis. IGFII can be increased as a consequence of genetic abnormalities and polymorphisms, and through epigenetic mechanisms. Decreasing IGFII levels thus can benefit human health. Degradation of IGFII is mediated by the insulin-like growth factor type 2 receptor (IGF2R). The growth-stimulatory effects of IGFII, and their attenuation by the IGF2R, are considered important for the evolution of IGFII/IGF2R interaction and imprinting. The IGFII/IGF2R interactions during development have been previously examined in mice carrying knock-out alleles of these genes or their regulators. Here we tested the ability of the IGF2R to ameliorate the negative effects of IGFII on development and survival in crosses between Igf2 and Igf2r transgenic mice, which may be a better model for natural variations in the levels of these genes' products. A fraction of hemizygous Igf2 transgenic mice die in the perinatal period, some with cleft palates, with an ensuing reduction in the frequency of transgenic mice among the surviving offspring. The Igf2r transgene lowers the frequency of cleft palate and increases the percentage of Igf2 transgenic mice among the live offspring. These findings draw attention to the fact that Igf2-associated lethality selects for the retention of IGFII/IGF2R binding in present day mammals; it may have played a similar role in the acquisition of IGFII/IGF2R binding in ancient mammals.     

Deletion of Selenoprotein M Leads to Obesity without Cognitive Deficits

Selenium is an essential trace element that is co-translationally incorporated into selenoproteins in the form of the 21st amino acid, selenocysteine. This class of proteins largely functions in oxidation-reduction reactions and is critically involved in maintaining proper redox balance essential to health. Selenoprotein M (SelM) is a thioredoxin-like endoplasmic reticulum-resident protein that is highly expressed in the brain and possesses neuroprotective properties. In this study, we first assessed the regional pattern of SelM expression in the mouse brain to provide insights into the potential functional implications of this protein in physiology and behavior. Next, we generated transgenic mice with a targeted deletion of the SelM gene and subjected them to a battery of neurobehavioral tests to evaluate motor coordination, locomotion, and cognitive function in comparison with wild-type controls. Finally, these mice were tested for several measures of metabolic function and body composition. Our results show that SelM knock-out (KO) mice display no deficits in measures of motor coordination and cognitive function but exhibit increased weight gain, elevated white adipose tissue deposition, and diminished hypothalamic leptin sensitivity. These findings suggest that SelM plays an important role in the regulation of body weight and energy metabolism.     

Utility of genetically modified mice for understanding the neurobiology of substance use disorders

Advances in our ability to modify the mouse genome have enhanced our understanding of the genetic and neurobiological mechanisms contributing to addiction-related behaviors underlying substance use and abuse. These experimentally induced manipulations permit greater spatial and temporal specificity for modification of gene expression within specific cellular populations and during select developmental time periods. In this review, we consider the current mouse genetic model systems that have been employed to understand aspects of addiction and highlight significant conceptual advances achieved related to substance use and abuse. The mouse models reviewed herein include conventional knock-out and knock-in, conditional knockout, transgenic, inducible transgenic, mice suitable for optogenetic control of discrete neuronal populations, and phenotype-selected mice. By establishing a reciprocal investigatory relationship between genetic findings in humans and genomic manipulations in mice, a far better understanding of the discrete neuromechanisms underlying addiction can be achieved, which is likely to provide a strong foundation for developing and validating novel therapeutics for the treatment of substance abuse disorders.     

The Use of Genetic “Knockout” Mice in Behavioral Endocrinology Research

The production of mice with specific deletion of targeted genes (knockouts) has provided a useful tool in understanding the mechanisms underlying behavior. There are many opportunities with this new tool for behavioral neuroendocrinology, specifically, and behavioral biology, generally. Although this genetic technique offers new opportunities to study the mechanisms of behavior, as with all behavioral techniques there are some potential limitations. For example, the products of many genes are essential to normal function, and inactivating the gene may prove lethal or induce gross morphological or physiological abnormalities that can complicate interpretation of discrete behavioral effects. Unexpected compensatory or redundancy mechanisms might be activated when a gene is missing and cloud interpretation of the normal contribution of the gene to behavior. Behavioral tests study the effects of themissinggene (and gene product), not the effects of the gene directly. This conceptual shortcoming can be overcome in the same way that it is overcome in other types of ablation studies, by collecting converging evidence using a variety of pharmacological, lesion, and genetic manipulations. Finally, because mammalian genome mapping is currently focused on mice (Mus musculus), standardized behavioral testing of mice should be adopted. Against those disadvantages are several important advantages to using knockout mice in behavioral research: (1) disabling a gene is often a very precise and “clean” ablation, (2) the effects of the gene product can be abolished without the side-effects of drugs, and (3) genetic manipulations may be the only way to determine the precise role of many endogenous factors on behavior. The use of new inducible knockouts, in which the timing and placement of the targeted gene disruption can be controlled, will be an extremely important tool in behavioral endocrinology research.     

Spontaneous colitis occurrence in transgenic mice with altered B7-mediated costimulation

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.     

Increased corticosterone levels in mice subjected to the rat exposure test

In recent years, there has been a notable interest in studying prey-predator relationships to develop rodent-based models for the neurobehavioral aspects of stress and emotion. However, despite the growing use of transgenic mice and results showing important differences in the behavioral responses of rats and mice, little research has been conducted regarding the responses of mice to predators. The rat exposure test (RET), a recently developed and behaviorally validated prey-predator (mouse-rat)-based model, has proven to be a useful tool in evaluating the defensive responses of mice facing rats. To further validate the RET, we investigated the endocrine and behavioral responses of mice exposed to this apparatus. We first constructed a plasma corticosterone secretion curve in mice exposed to a rat or to an empty cage (control). Rat-exposed mice showed a pronounced rise in corticosterone levels that peaked 15 min from the beginning of the predator exposure. The corticosterone levels and behavioral responses of mice exposed to a rat or to a toy in the RET apparatus were then measured. We observed high plasma corticosterone levels along with clear avoidance behaviors represented by decreases in tunnel and surface area exploration and increases in risk assessment behaviors and freezing. This strongly suggests that the test elicits a repertoire of behavioral responses compatible with an aversion state and indicates that it is a promising model for the evaluation of prey-predator interactions. However, more physiological, neurochemical, and pharmacological studies are needed to further validate the test.